Beschreibung:
<jats:p>The extent and nature of genetic differentiation in <jats:styled-content style="fixed-case"><jats:italic>Semotilus atromaculatus</jats:italic></jats:styled-content>, one of the most abundant and widespread leuciscids in North America, were evaluated based on mitochondrial (mt) and nuclear DNA sequence variation. Phylogenetic relationships were first inferred based on a fragment of the cytochrome b (<jats:italic>cytb</jats:italic>) region and the nuclear intron <jats:italic>s7</jats:italic> gene for <jats:italic>S. atromaculatus</jats:italic> and all other congeners as well as representative species from all other genera in the creek chub–plagopterin clade. The phylogeography of major haplogroups of <jats:styled-content style="fixed-case"><jats:italic>S. atromaculatus</jats:italic></jats:styled-content> was also assessed according to variation in a fragment of the mitochondrial <jats:italic>cytb</jats:italic> region from 567 individuals across its range. All analyses identified <jats:styled-content style="fixed-case"><jats:italic>S. thoreauianus</jats:italic></jats:styled-content>, <jats:italic>S. lumbee</jats:italic> and <jats:styled-content style="fixed-case"><jats:italic>S. corporalis</jats:italic></jats:styled-content> as reciprocally monophyletic groups. Analyses of nuclear sequence variation resolved <jats:styled-content style="fixed-case"><jats:italic>S. atromaculatus</jats:italic></jats:styled-content> as a single clade, where <jats:italic>S. thoreauianus</jats:italic> and <jats:styled-content style="fixed-case"><jats:italic>S. lumbee</jats:italic></jats:styled-content> were recovered as the sister group to <jats:italic>S. atromaculatus</jats:italic>, and <jats:styled-content style="fixed-case"><jats:italic>S. corporalis</jats:italic></jats:styled-content> was resolved as sister to all other species in the genus. Analyses of mtDNA sequence variation recovered <jats:styled-content style="fixed-case"><jats:italic>S. atromaculatus</jats:italic></jats:styled-content> as three well supported and differentiated monophyletic groups, with a widespread genetically homogeneous lineage extending across most of the current range of the species; a more geographically restricted and geographically structured lineage in the southern Appalachians, sister group to <jats:styled-content style="fixed-case"><jats:italic>S. lumbee</jats:italic></jats:styled-content>; and a geographically restricted lineage was identified from two Gulf Slope basins. Evidence of complex mito‐nuclear discordance and phylogeographic differentiation within <jats:styled-content style="fixed-case"><jats:italic>S. atromaculatus</jats:italic></jats:styled-content> illustrates that further analysis of widespread species is warranted to understand North American freshwater fish diversity and distributions.</jats:p>